A number of studies have shown that the flaviviruses West Nile virus (WNV) and dengue (DENV) infection inhibits the activation of the interferon(IFN) signaling cascade, thus blocking antiviral responses. Our investigations have shown that WNV blocks IFN signaling cascade by inducing a decrease in an IFNreceptor subunit, IFNAR1. Further, studies in our lab and others have shown that the WNV nonstructural protein NS4B is a determinant in blocking IFN responses. Individually expressed DENV NS4B has also been shown to inhibit IFN signaling. However, the mechanism DENV employs to inhibit IFN responses is unclear. The purpose of the proposed research program is to investigate the mechanism responsible for DENV inhibition of the IFN response through three specific aims. In the first aim, we will use a biochemical approach to analyze the stability and activation status of proteins that participate in the IFN signaling pathway. Specifically, we will determine if viral proteins inhibit the IFN response through direct modification or decrease of cellular proteins or their association with IFN-D receptor complex. Experiments were designed to differentiate between direct and indirect mechanisms of antagonism. With the second aim, we will use shRNA technology to interrogate the IFN-stimulated genes (ISG) that establish antiviral environments in cells. Our goal is to determine the specific proteins that inhibit DENV replication. In the third aim, we will use a genetic approach to identify the viral protein(s) responsible for the inhibition of the IFN response in DENV-infected cells. Our approach will employ subtle mutagenesis in the coding region of an infectious clone and screen large numbers of viruses through a cell based assay. Our goal is to identify replication competent DENV mutants that cannot inhibit IFN signaling. DENV is a major global pathogen of significant medical and economic concern. To date, no vaccine or therapeutic treatments exist. The studies proposed here will provide better understanding of DENV activities that modulate innate immune responses as well as a clearer picture of the cellular defenses employed against infection. The information and reagents gathered can be used to develop more effective antiviral therapies.